This application is in response to NCI Provocative Question (PQ) 9 to test an innovative hypothesis that senescent cells (SCs) induced by ionizing radiation (IR), particularly senescent type 2 alveolar epithelial cells (AEC2s), are the perpetuators primarily responsible for initiating pathogenesis and driving progression of radiation-induced pulmonary fibrosis (RIPF); therefore, clearance of SCs with a senolytic drug that selectively kills SCs can prevent RIPF and/or delay its progression and, more importantly, reverse existing tissue fibrosis, even after RIPF becomes a progressive disease. This hypothesis is based on the long-term understanding of tissue fibrosis pathogenesis and the exciting new findings about the role of SCs in pulmonary fibrosis, including RIPF. Specifically, pulmonary fibrosis is a disease caused by aberrant wound healing responses that are presumably initiated by cellular senescence induction in AEC2s, which are considered alveolar stem cells (ASCs). This process causes AEC2 defects in self-renewal and differentiation and an inability to generate AECs for maintenance of the alveolar epithelial barrier and to repair damaged lung epithelium. Senescent AEC2s also produce increased levels of reactive oxygen species and release an array of proinflammatory and profibrotic factors, as a result of senescence-associated secretory phenotype (SASP) expression, which may initiate and perpetuate pulmonary fibrosis development by inducing chronic oxidative stress and inflammation and promoting abnormal repair of tissue injury. This is supported by the findings that (1) AEC2 senescence, induced by AEC2-specific deletion of telomeric repeat-binding factor 2 (Trf2), in adult mice causes ASC failure, provokes pulmonary inflammation and fibrotic responses, and sensitizes mice to bleomycin- induced pulmonary fibrosis and (2) cellular senescence inhibition can prevent RIPF and delay bleomycin-induced pulmonary fibrosis progression. Our preliminary studies showed that treatment of the thoracic irradiated mice with ABT-263, a specific Bcl-2/xl inhibitor and a newly discovered senolytic drug by our group, almost completely reversed RIPF, even when ABT-263 treatment was delayed until RIPF was established. To our knowledge, this is the first study to demonstrate that RIPF can be reversed by a drug after it becomes a progressive disease. From these novel findings, we will test our hypothesis through the following specific aims using our well- established thoracic irradiation mouse model: (1) validate that SCs are the perpetuators primarily responsible for initiating pathogenesis and driving progression of RIPF; (2) elucidate the mechanisms by which clearance of SCs prevents and reverses RIPF; and (3) develop a safer and more effective senolytic drug treatment for RIPF. RIPF is one of the most deleterious late effects of thoracic radiotherapy. Our proposed studies will lead to the identification of the underlying mechanisms whereby IR causes pulmonary fibrosis and development of an innovative mechanism-based therapy that can not only prevent and/or delay RIPF but also reverse RIPF.